1. Field of the Invention
The present invention relates to trehalose and its production and use, in particular, to trehalose or saccharide containing the same which is obtainable by cultivating in a nutrient culture medium with maltose a microorganism capable of producing maltose/trehalose conversion enzyme, as well as to a process to produce the same and use thereof.
2. Description of the Prior Art
Trehalose (alpha,alpha-trehalose) has been known from ancient times as non-reducing saccharide composed of glucose, and as described in Advances in Carbohydrate Chemistry, published by Academic Press Inc., New York, N.Y., USA, Vol.18, pp.201-225 (1963) and Applied and Environmental Microbiology, Vol.56, pp.3,213-3,215 (1990), its trace but extensive distribution is found in microorganisms, mushrooms and insects. Since as is trehalose, non-reducing saccharides cause no aminocarbonyl reactions with substances bearing amino groups such as amino acids and proteins and therefore neither deteriorate nor alter them, the saccharides have been deemed to be useful in utilizing and processing such substances with no fears of their browning and deterioration: Thus establishment of processes which would enable their industrial-scale production has been in great expectation.
There have been known several processes to produce trehalose, for example, those using microorganism cells as disclosed in Japanese Patent Kokai No.154,485/75 and those converting maltose by combination of maltose phosphorylase and trehalose phosphorylase as disclosed in Japanese Patent Kokai No.216,695/83. The former process using microorganism cells is however inadequate for industrial-scale process because the trehalose content in microorganism cells as starting material is generally low, i.e. less than 15 w/w % (the percentages appeared hereinafter mean "w/w %" unless specified otherwise), and the extraction and purification steps for trehalose are very complicated. While the latter process using maltose phosphorylase and trehalose phosphorylase has not been realized in industrial scale due to the demerits that both enzymes commonly act via glucose-1-phosphate and this hinders elevated concentrations for substrates, that the yield for trehalose is low because both enzymes irreversibly act in the same reaction system, and further that such reaction system is very difficult to stably maintain and smoothly proceed.
In connection with this, Gekkan Food Chemical (Monthly Food Chemical), "Recent Aspects and Issues in Utilization and Development of Starch", August, pp.67-72 (1992) comments in the corner of "Oligosaccharides" that although trehalose would have very extensive uses, its enzymatic production using any direct saccharide-transferring or hydrolyzing reactions has been deemed to be scientifically impossible at the present time, confirming that the production of trehalose from starch as material using enzymatic reactions has been deemed to be scientifically impossible.
While it is known that partial starch hydrolysates, for example, liquefied starch, dextrins and maltooligosaccharides which are all produced from starch, generally exhibit reducing powers due to the reducing end groups in their molecules. Such a partial starch hydrolysate will be designated as "reducing partial starch hydrolysate" in this specification. The reducing powers of reducing partial starch hydrolysates on dry solid basis are usually expressed by "Dextrose Equivalent" or "DE". Also is known that reducing partial starch hydrolysates with higher DE values, which are generally small molecules, exhibit low viscosities and strong sweetening powers, as well as high reactivities to substances with amino groups such as amino acids and proteins, which cause the aminocarbonyl reaction leading to browning, unpleasant smell and deterioration.
The characteristics of reducing partial starch hydrolysates vary dependently on the magnitudes of their DE and therefore the relationship between particular reducing partial starch hydrolysates and their DE values is very important. It has been however believed in the art to be impossible to cut off this relationship.
The sole method to cut off the relationship is to change reducing partial starch hydrolysates into non-reducing saccharides, for example, by converting their reducing groups into alcohol groups by high-pressure hydrogenation. This method however needs high-pressure autoclaves, safety facilities and careful control to prevent disasters, as well as consuming large amounts of hydrogen and energy. Further the obtained saccharide alcohols differ from reducing partial starch hydrolysates in the point that reducing partial starch hydrolysates consist of glucose moieties, while the saccharide alcohols consist of glucose and sorbitol and this may cause transient indigestion and diarrhea. Thus it has been in great demand to establish any methods by which the reducing powers of reducing partial starch hydrolysates are decreased or even eliminated without changing glucose moieties which compose reducing starch hydrolysates.
To solve these, the present inventors disclose in Japanese Patent Application No.144,092/94 a novel enzyme (referred to as "maltose/trehalose conversion enzyme" hereinafter) which is capable of converting maltose into trehalose, thus establishing a process to produce trehalose or saccharide containing the same from maltose using this maltose/trehalose conversion enzyme.
It was however found later that, unfavorably, such a process needed an extended time for cultivation of microorganisms to produce enzymes, recovery of the enzymes and enzymatic reaction to convert maltose into trehalose. Thus it is in great demand to establish any processes which would be more readily feasible in the production of trehalose from maltose with less complicated steps including those for preparation of enzymes.